The cholesterol biosynthesis enzyme FAXDC2 couples Wnt/β-catenin to RTK/MAPK signaling

Wnts, cholesterol, and MAPK signaling are essential for development and adult homeostasis. Here, we report that fatty acid hydroxylase domain containing 2 (FAXDC2), a previously uncharacterized enzyme, functions as a methyl sterol oxidase catalyzing C4 demethylation in the Kandutsch-Russell branch of the cholesterol biosynthesis pathway. FAXDC2, a paralog of MSMO1, regulated the abundance of the specific C4-methyl sterols lophenol and dihydro-T-MAS. Highlighting its clinical relevance, FAXDC2 was repressed in Wnt/β-catenin–high cancer xenografts, in a mouse genetic model of Wnt activation, and in human colorectal cancers. Moreover, in primary human colorectal cancers, the sterol lophenol, regulated by FAXDC2, accumulated in the cancerous tissues and not in adjacent normal tissues. FAXDC2 linked Wnts to RTK/MAPK signaling. Wnt inhibition drove increased recycling of RTKs and activation of the MAPK pathway, and this required FAXDC2. Blocking Wnt signaling in Wnt-high cancers caused both differentiation and senescence; and this was prevented by knockout of FAXDC2. Our data show the integration of 3 ancient pathways, Wnts, cholesterol synthesis, and RTK/MAPK signaling, in cellular proliferation and differentiation.


Animal studies
Our study examined male and female animals, and similar findings are reported for both sexes.Mouse xenograft models were established by orthotopic injection of HPAF-II or AsPC-1 cells in NSG mice as described previously (4).For tumor growth studies, HPAF-II, FAXDC2 knockout, FAXDC2 overexpressing (OE) HPAF-II, HCT116 or HT29 cells were suspended in 50% matrigel and injected subcutaneously into the flanks of NSG mice.Mice were treated with ETC-159 after the establishment of tumors.ETC-159 was formulated in 50% PEG 400 (vol/vol) in water and administered by oral gavage at a dosing volume of 10 μL/g body weight (4).The tumor dimensions were measured with a caliper routinely, and the tumor volumes were calculated as 0.5 x length x width 2 (5).All mice were sacrificed 8 hours after the last dose.At sacrifice, tumors were resected, weighed, and snap frozen in liquid nitrogen or fixed in 10% neutral buffered formalin.

RNA Isolation and qRT-PCR
Tumors were homogenized in RLT buffer using a polytron homogenizer, and total RNA was isolated using an RNAeasy kit (Qiagen, Germany) according to the manufacturer's protocol.The RNA-seq libraries were prepared using the Illumina TruSeq stranded Total RNA protocol with subsequent PolyA enrichment.For qRT-PCR, RNA was reverse transcribed with iScript reverse transcriptase (BioRAD, Hercules, CA, USA).Real-time quantitative PCR (qPCR) was performed using SsoFast™ EvaGreen® assay Supermix from BioRad (Hercules, CA, USA).EPN1 and ACTB were used as housekeeping genes.The primers used are listed in Table S3.

Immunohistochemistry
Tissue sections of formalin-fixed paraffin-embedded tumors were deparaffinized in xylene and rehydrated using an ethanol gradient.After antigen retrieval with sodium citrate buffer pH 6.0 for 10 min, the endogenous peroxidase activity was blocked by incubation with H2O2.The sections were then incubated overnight with 1:200 diluted phospho-p44/42 MAPK antibody (4376S, Cell Signaling Technologies, Danvers, MA) followed by HRP conjugated anti-rabbit secondary antibody for 1 h.Incubation with 3,3'-diaminobenzidine chromogen substrate resulted in brown staining of phospho-ERK positive cells, and the nuclei were counterstained with Mayer's hematoxylin.Brightfield images were acquired on a Nikon Eclipse Ni-E microscope.OCT-embedded tissue sections were stained for SA-β-galactosidase using the protocol described previously (2).

Sterol extraction and GC/MS analysis for sterol identification
To extract the sterols, the tumor fragments were saponified with 1 ml of 10% methanolic KOH at 80°C for two hours, followed by extraction with 1 ml n-hexane.One ug of epicoprostanol (0.1 mg/ml in toluene) was added as an internal standard.The sterols in the mixture were extracted with 1 ml of n-hexane and dried under nitrogen.The residues were stored in a CaCl2 desiccator for 24 h to remove moisture.The extracted sterols were then converted to their trimethyl silane esters using N, O-Bis (trimethylsilyl), trifluoroacetamide (BSTFA) with trimethylchlorosilane (TMCS) (99:1), and pyridine as a catalyst.For analysis, each derivatized sample (5 μl) was injected into the 0.25 mm capillary column of GC/MSD (Agilent GC 6890, fitted with an automatic Liquid Sampler and a 5973 quadrupole MS detector (Agilent Technologies).The inlet temperature was 250˚C, and the helium carrier gas flowed at a constant rate of 1.2 mL/min.The GC oven temperature was set at 170˚C initially for 1 min with an increase to 280˚C at a rate of 40˚C min-1 and then held for 20 min.The transfer line temperature was set at 280˚C, MS source at 230˚C, and the quadrupole temperature at 150˚C.Ionization was by electron impact at 70 eV.The mass calibrant perfluorotributylamine was used to auto-tune the MSD.The MSD was run at scan mode ranging from 50 to 550 amu.
The data was analyzed using Agilent GC/MSD Productivity Chemstation software and Automated Mass Spectral Deconvolution and Identification System (AMDIS).The sterols were identified by comparing their retention times and mass spectra to the authentic standards and commercial GC/MS database (NIST/EPA/NIH Mass Spectral Library, NIST 08).The sterols were identified by comparing their retention times and mass spectra to the authentic standards, commercial GC/MS database (NIST/EPA/NIH Mass Spectral Library, NIST 08), or purified 4,4-dimethylcholesta-8,24-dienol (isolated from the Saccharomyces cerevisiae ERG25 mutant (6), Figure S5E).The database has no sample for 4,4-dimethylcholest-8-enol, and no standard was available.However, the mass spectrum of this sterol was similar to 4,4-dimetylcholesta-8,24-dienol but two mass units higher, and it eluted about 1.5 min early, indicating that it has a saturated side chain.The strong m/z=135 was from the 4,4-dimethyl substitution.The mass spectrum of the targeted sterol showed high similarity to that of the published mass spectrum of 4,4-dimethylcholest-8-enol (7), and it was confirmed to be 4,4dimethylcholest-8-enol.

Semi-quantitative analysis of sterols
The selective ion monitoring (SIM) method was developed to quantify the trace amount of sterols in the tumor tissues with high sensitivity (detection limit of 10 ng on column).The first group of ions was from 9 min to 14.6 min, and m/z 355 amu for the internal standard was monitored with 25% dwell time; the second group of ions was from 14.6 min to 28 min with m/z 458 amu for cholesterol and lathosterol, m/z 472 for campesterol and lophenol, m/z 484 for 4,4-dimethylcholesta-8,24-dienol and m/z 486 for 4,4-dimethylcholest-8-enol and sitosterol.Each ion was monitored with a 25% dwell time.The intensity of each ion was integrated using the Chemstation software, and TIC (total ion current), which represents the amount of each sterol, was calculated by the conversion factor of the SIM to TIC derived from the standards (the conversion factor for 4,4-dimethylcholesta-8,24-dienol was used for the calculation of 4,4-dimethylcholest-8-enol since their mass spectra were very similar).The amount of each sterol was normalized to the internal standard and the fresh tissue weight.The amount of cholesterol could not be accurately determined because of its high concentration in the tissue, and the mass detector was saturated even in SIM mode.

RNA-seq analysis
Data processing and QC: RNA-seq datasets were analyzed and clustered as described in Madan et al. (4).The RNA-seq of FAXDC2 KO tumors was performed using the same pipeline.Sequences were assessed for quality, and reads from mouse (mm10) were removed using Xenome (8).The remaining reads were aligned against hg38 (Ensembl version 79) using STAR v2.5.2 (9) and quantified using RSEM v1.2.31 (10).Reads mapping to chrM or annotated as rRNA, snoRNA, and snRNA were removed.Genes with less than ten reads mapping on an average were removed.Differential expression analysis was performed using DESeq2 (11).Independent filtering was not used in this analysis.Pairwise comparisons were performed using a Wald test.To control for false positives due to multiple comparisons in the genome-wide differential expression analysis, we used the false discovery rate (FDR) computed using the Benjamini-Hochberg procedure.Gene-level counts were transformed using a variance-stabilizing transformation and converted to z-scores.Time was transformed using a square root transformation.All genes differentially expressed over time (DESeq2, false discovery rate (FDR) < 10%) were clustered using GPClust (12) using the Matern32 kernel with a concentration (alpha) parameter of 0.001 and a length scale of 6.5.
Fold changes for all FAXDC2-dependent genes (interaction test, FDR < 10%) were clustered using hierarchical clustering with complete linkage and correlation distance.We used the average silhouette width to determine this dataset's optimal number of clusters (Figure S8, N=6 tumors/group).

Functional enrichment analysis
For the analysis of the Wnt-repressed genes (Figure 1), Gene Ontology (GO) enrichments were performed using GOStats (13) using all genes differentially expressed (FDR < 10%) as background.ReactomePA (14) was used for investigating pathway enrichments using the same background.Terms with an FDR < 10% were defined as significantly enriched.For the analysis of the FAXDC2dependent genes (Figure 8), functional enrichments were performed using all expressed genes as background.

Transcription factor binding sites (TFBS) analysis
TFBS motifs were obtained from the JASPAR2018 database (15).Promoters were defined as 500 bp upstream and downstream from the ENSEMBL annotated transcription start site.AME was used to search for enriched motifs in these regions using all expressed genes not in a specific cluster as background and a hit-lo-fraction of 0.5.p-values reported by AME were corrected for multiple testing using FDR (16).Motifs with an FDR < 10% were defined as significantly enriched.A complete list of the motif enrichments for the Wnt-repressed clusters C2, C4, C6, and C8 is reported in Supplemental Table S4.A complete list of motif enrichments for clusters of genes identified as being FAXDC2-dependent is reported in Supplemental Table S2.
Biorender was used to draw the graphical abstract.

A. Replot of heat map of Wnt regulated genes in HPAF-II orthotopic xenografts: Mice bearing
HPAF-II orthotopic tumors were treated with a Wnt inhibitor (ETC-159, 37.5 mg twice daily).Tumors were resected at indicated time points after the treatment.RNA sequencing was performed to measure the transcript abundance.Based on the temporal dynamics of the transcriptional response to Wnt inhibition, these genes were distributed amongst 17 clusters.

B. Expression of multiple cholesterol biosynthesis pathway genes is Wnt/β-catenin dependent.
Expression of indicated sterol pathway genes in HPAF-II orthotopic xenografts with or without stabilized β-catenin from mice treated with vehicle or ETC-159 for 56 hours was analyzed by RNA sequencing.Data shows the relative gene expression in the four groups.N=3-5 mice/group.

C-D. TCF7L2 knockout increases Axin2 expression:
TCF7L2 knockout HT-29 and HCT116 colon cancer xenografts had lower levels of AXIN2 compared to the WT controls.P values were calculated using the Mann-Whitney U test.

E-F. SREBP knockdown does not alter Wnt-regulated FAXDC2 expression. HPAF-II cells were
transfected with multiple siRNAs against SREBP1 or SREBP2 for 24 h, followed by treatment with ETC-159 for 72 h.Expression of FAXDC2 and SREBP was measured by qRT-PCR. A.

Figure S3
Figure S3: Identification of methyl sterols using GC-MS (accompanying Figure 2) A. Representative GC trace of total sterols recovered from a representative HPAF-II tumor.
Internal standard (IS) epicoprostanol (3α-inverted C-OH and A/B cis ring generating a bent 190 ring structure affording a sterol standard that elutes before cholesterol and does not interfere with the sterol analysis of natural metabolites) and sterols identified in the chromatogram are indicated.

B.
Mass spectra of TMS-derivatized sterols isolated from tumors and corresponding spectra of standards.Mass spectra in the left column are from tumor samples, the middle column is from 195 the NIST library (red) or from (6) (black), and the right column (blue) from the Nes sterol collection.

B. FAXDC2 knockout blunts the Wnt-inhibition-mediated increase in cellular differentiation.
The expression of differentiation-associated genes was analyzed in the tumors of all four groups.Each data point represents an individual tumor.Hypergeometric test, FDR<10%.

B.
Antibody control for figure 2D.HeLa cells were co-transfected with vectors encoding epitopetagged constructs of MSMO1, NSDHL, and FAXDC2.Cells visualized after staining with fluorescence-tagged secondary antibodies show no background staining.

5 .Figure S7 (accompanying Figure 6 )A.
Figure S4A.FAXDC2 is repressed in primary colorectal cancers compared to adjacent normal tissues:Expression of FAXDC2 was compared in primary cancers and adjacent normal tissues by qRT-PCR.P values were calculated using the Mann-Whitney test.B-C.FAXDC KO clones obtained usingCRISPR methodology show the deletion of large regions of Exon 5.The genomic sequence of FAXDC2 knockout clone 3 (A) and clone 12 (B) both show deletion of a large region of Exon 5. D. Loss of FAXDC2 protein in the FAXDC2 KO xenografts.Protein lysates from the parental HPAF-II and FAXDC2 KO xenografts from the vehicle or ETC-159 treated mice were probed with the FAXDC2 or GAPDH antibody.Each lane represents tumor lysate from an individual mouse.
Figure S8 Cre Rnf43 fl/fl /Znrf3 fl/fl mice with activated Wnt signaling have increased pancreatic weight compared to the WT mice.Treatment with ETC-159 for 21 days reduces this increase in pancreatic weight.Each dot represents an individual mouse.Data is from two independent biological experiments with 5-6 mice/group.P values were calculated using Mann-Whitney test.